Therefore, intensive research has been aimed at understanding the mechanisms of cytoadherence, so that therapeutic agents blocking these interactions might eventually be designed (7, 8)

Therefore, intensive research has been aimed at understanding the mechanisms of cytoadherence, so that therapeutic agents blocking these interactions might eventually be designed (7, 8). The importance of electron-dense structures (knobs) on the surface of the parasitized red blood cell to cytoadherence has long been recognized. red blood cells to the endothelial lining RU.521 (RU320521) of capillaries and venules of various tissues, including the brain. Ligands on the surface of parasitized red cells can bind to a number of endothelial cell receptors, including CD36 (1), intercellular adhesion molecule-1 (2), thrombospondin (3), chondroitin-4-sulfate (4), vascular cell adhesion molecule-1 (5), E selectin (5), and platelet/endothelial cell adhesion molecule-1 (6). Therefore, intensive research has been aimed at understanding the mechanisms of cytoadherence, so that therapeutic agents blocking these interactions might eventually be designed (7, 8). The importance of electron-dense structures (knobs) on the surface of the parasitized red blood cell to cytoadherence has long been recognized. A major constituent of knobs is the knob-associated histidine-rich protein (KAHRP) (9), localized under the red cell membrane. During culture, some lines of lose the ability to produce knobs (10), and they generally lose the ability to cytoadhere. This is a consequence of subtelomeric deletions of the region of chromosome 2 bearing the KAHRP gene RU.521 (RU320521) (11). It is important to note, however, that there are exceptions. Clone B8, for example, can adhere to melanoma cells, although it is KAHRP-negative and knob-negative (12). Recently, a targeted recombinational knockout of the KAHRP gene has been used to demonstrate RU.521 (RU320521) that KAHRP itself is essential for knobs and stable cytoadherence under physiological shear-stress levels (13). erythrocyte membrane protein 1 (PfEMP1) is a variable molecule of approximately 250 kDa located on the surface of the parasitized red blood cell (14). PfEMP1 is now used as a collective term for any product of the multigene family. It is clear that the parasite can undergo clonal antigenic variation by switching on the expression of different members of this set of about 50 polymorphic genes (15C17). Because switching can occur at up to 2% per generation in some instances (18), clonal parasite populations can express a mixture of PfEMP1 types even though only one (or at most a few) is expressed per cell. The PfEMP1 type expressed has an important role in determining the receptor specificity of the parasitized red blood cell (19C22). Nevertheless, at least one more previously undefined gene product is also essential for cytoadherence. During cultivation, isolates of commonly undergo loss of cytoadherence, as measured by binding to C32 melanoma cells (10). We have associated this loss with subtelomeric deletions of chromosome 9 (23C25), where the independent deletion breakpoints are tightly clustered (26). Mixed parasite populations arise LY6E antibody during propagation of clones because of such deletions. Binding RU.521 (RU320521) these populations to melanoma cells resulted in the selection of parasites with the undeleted form of chromosome 9 in all lines tested (24, 27). We proposed that a gene essential for cytoadherence must be located in this region (24). Clone ItG2 differs from a number of other parasite lines in that cytoadherence is stable over many generations (28), so it has been used to study cytoadherence in several laboratories. Studies (25) have shown that cytoadherent clones derived from ItG2 (e.g., B8) possess a chromosome 9 of intermediate size between that of cytoadherent isolate 1776 and its noncytoadherent derivative clone C10 (23). This is because of a deletion of intermediate size at the right end of the chromosome, as well as an internal deletion of about 15 kb that deletes an ORF at the site of the most common breakpoints in other isolates (26). The remaining segment of about 55 kb in ItG2 is colinear with its counterpart in the widely used stably cytoadherent clone of genes are detectable in this region (26), it must contain a unique cytoadherence gene. We describe here the identification of a gene from this locus that is required for cytoadherence to C32 melanoma cells and CD36. The gene has been named the cytoadherence-linked asexual gene ((bases 313C1,021, GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AF055476″,”term_id”:”3694804″,”term_text”:”AF055476″AF055476), containing neither the 5 nor 3 end of the gene (Fig. ?(Fig.1).1). This PCR product was blunt-end cloned into the unique fragment was inserted were sequenced to confirm the orientation with respect to the drug resistance cassette. Only clones that had the insert in the same orientation as the drug resistance cassette were used. This vector was named pAC4-Clag9. Open in a separate window Figure 1 (gene incorporated into the vector pAC4-Clag9. ( ) Region of gene 5 and 3 to that incorporated into the vector pAC4-Clag9. (calmodulin 5 untranslated region. () dihydrofolate reductase, thymidylate synthase, gene. (?) histidine-rich protein two 3 untranslated.